Evaluation of truncated NhhA protein as a candidate meningococcal vaccine antigen.

NhhA (Neisseria hia homologue) is an outer membrane protein from Neisseria meningitidis, the causative agent of meningococcal disease. The protein is surface exposed and its expression in a wide range of meningococcal strains suggests it is a promising vaccine candidate. In addition, immunization of mice with outer membrane vesicles of strains that overexpress NhhA in conjunction with one of TbpA, Omp85 or NspA results in synergistic bactericidal responses. We previously showed that the NhhA sequence is highly conserved between strains, with the majority of the differences localized to four distinct variable regions located in the amino-terminal region of the mature protein. In this study, N. meningitidis strains were constructed that over-express wild-type NhhA. Strains expressing truncated versions of NhhA, with deletions from the amino-terminal region that removed the most variable regions, were also made. These expression strains were also modified so that immunodominant, phase- and antigenically-variable outer membrane proteins were not expressed, truncated lipooligosaccharide (LOS) expression was genetically fixed (no phase variability), and capsular polysaccharide expression abolished. Outer membrane vesicles derived from these strains were used to immunize mice. As previously observed, a synergistic effect involving another antigen, TbpA, was required to demonstrate bactericidal activity. The highest bactericidal response against a heterologous strain was obtained with a truncated variant of NhhA. These results indicate that removal of (a) variable region(s) does not reduce bactericidal responses against NhhA, and that bactericidal targets exist in regions other than the variable N-teminus. This provides the basis for future examination of responses against truncated NhhA in protecting against heterologous NhhA strains, and further evaluation of truncated NhhA as a candidate for inclusion in a vaccine against all serogroups of N. meningitidis.

An outer membrane receptor of Neisseria meningitidis involved in zinc acquisition with vaccine potential.

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.

Additive and synergistic bactericidal activity of antibodies directed against minor outer membrane proteins of Neisseria meningitidis.

Neisseria meningitidis serogroup B is a major cause of bacterial meningitis in younger populations. The available vaccines are based on outer membrane vesicles obtained from wild-type strains. In children less than 2 years old they confer protection only against strains expressing homologous PorA, a major, variable outer membrane protein (OMP). We genetically modified a strain in order to eliminate PorA and to overproduce one or several minor and conserved OMPs. Using a mouse model mimicking children's PorA-specific bactericidal activity, it was demonstrated that overproduction of more than one minor OMP is required to elicit antibodies able to induce complement-mediated killing of strains expressing heterologous PorA. It is concluded that a critical density of bactericidal antibodies needs to be reached at the surface of meningococci to induce complement-mediated killing. With minor OMPs, this threshold is reached when more than one antigen is targeted, and this allows cross-protection.

Shielding of immunogenic domains in Neisseria meningitidis FrpB (FetA) by the major variable region.

The meningococcal iron-limitation-inducible outer membrane protein FrpB (FetA) has been shown to induce bactericidal antibodies, and is, therefore, considered a vaccine candidate. However, these antibodies are strain specific and, consistently, epitope mapping showed that they are directed against a region, located in a surface-exposed loop, L5, that displays considerable sequence variability between strains. Here, we attempted to redirect the immune response to more conserved domains of the protein by deleting L5. Immunization with an FrpB protein lacking L5 resulted in a bactericidal antibody response, and epitope mapping showed that these antibodies were directed against loop L3, which also displays considerable sequence variability. To re-direct the immune response further, immunizations were performed with an FrpB protein lacking both L5 and L3. The antibodies obtained were not bactericidal. Furthermore, the bactericidal antibodies against L3 were only bactericidal in the absence of L5, and immunofluorescence microscopy experiments showed that L5 efficiently shields other immunogenic cell surface-exposed epitopes outside of this region on living cells. Whereas the ability of micro-organisms to vary surface-exposed domains that are targets for protective immunity has long been established, the current work shows that such domains can be remarkably efficient in shielding other, more conserved epitopes.

Immunogenicity and structural characterisation of an in vitro folded meningococcal siderophore receptor (FrpB, FetA).

The iron-limitation-inducible protein FrpB of Neisseria meningitidis is an outer-membrane-localized siderophore receptor. Because of its abundance and its capacity to elicit bactericidal antibodies, it is considered a vaccine candidate. Bactericidal antibodies against FrpB are, however, type-specific. Hence, an FrpB-based vaccine should comprise several FrpB variants to be capable of providing broad protection. To facilitate the development of a meningococcal subunit vaccine, we have established a procedure to obtain large quantities of the protein in a native-like conformation. The protein was expressed without its signal sequence in Escherichia coli, where it accumulated in inclusion bodies. After in vitro folding, the protein was biochemically, biophysically and biologically characterised. Our results show that in vitro folded FrpB assembles into oligomers, presumably dimers, and that it induces high levels of bactericidal antibodies in laboratory animals.

Vaccine potential of the Neisseria meningitidis lactoferrin-binding proteins LbpA and LbpB.

The vaccine potential of the Neisseria meningitidis lactoferrin-binding proteins LbpA and LbpB was evaluated. Sequencing and sequence alignments suggested that LbpA is relatively well conserved with variability largely restricted to two surface-exposed loops in a proposed topology model. Consistently, antisera raised against synthetic peptides corresponding to exposed loops generally recognized the LbpA proteins of many different strains. Hence, LbpA was considered an attractive vaccine candidate. LbpB shows a higher degree of sequence variability. For immunisation studies, LbpA was overproduced in N. meningitidis and a histidine-tagged LbpB protein was produced in Escherichia coli. Outer membrane vesicles carrying overproduced LbpA and purified LbpB were used to immunise laboratory animals. The bactericidal activity and cross-reactivity of the antibodies was evaluated using meningococcal strains of various clonal lineages. In addition, LbpB-specific monoclonal antibodies were analysed by Western blots and whole-cell enzyme-linked immunosorbent assays. Our results show that both proteins are immunogenic and able to induce bactericidal antibodies, but that the cross-reactivity of these antibodies is limited.